Molecular Phylogenetics And Evolution

410M.F. Medina et al. / Molecular Phylogenetics and Evolution 100 (2016) 409–423The semi-fossorial, African snake-eyed skink genus Panaspiscurrently includes eight savanna and lowland rainforest speciesdistributed throughout sub-Saharan Africa (Uetz and Hošek,2015). In the 20th century, the taxonomic composition of thegenus Panaspis was based on morphological characters, includingskull morphology, head scalation, and distinctive characters inthe lower eyelid (Broadley, 1989; Fuhn, 1969, 1972; Greer, 1974;Perret, 1973, 1975, 1982). As a result, some African and Eurasianskink species were moved back and forth between different scincidtaxa, including Ablepharus, Afroablepharus, Lacertaspis, Leptosiaphos,and Panaspis (Fuhn, 1969, 1970; Greer, 1974; Perret, 1973, 1975).The recurrent allocation of African savanna scincid speciesamong these closely related genera in the 20th century resultedfrom the disparate morphological work of several herpetologists.After the ablepharine (lower eyelid fused with the supercilium)and pre-ablepharine (lower eyelid not completely fused, forminga palpebral slit) eye conditions were discovered (Boulenger,1887), and Fuhn (1969) noted that skull morphology could be usedto delimit scincid taxa, the genus Panaspis was restricted to Africanspecies. Continued use of skull morphology also supported the separation of the family Scincidae into four subfamilies: Acontinae,Feylininae, Lygosominae, and Scincinae (Greer, 1970). Recentmolecular and morphological evidence (Hedges, 2014; Hedgesand Conn, 2012; Skinner et al., 2011) suggested skinks could bedivided into as many as nine families. Although considered controversial, ignored, or rejected by subsequent authors (e.g., Lambertet al., 2015; Linkem et al., in press; Pyron et al., 2013), this newsubdivision continues to support skinks as a monophyletic group(Hedges, 2014). Under a modified version of this classification,the genera Afroablepharus, Lacertaspis, Leptosiaphos, and Panaspisare allocated to the Subfamily Eugongylinae (Hedges, 2014; Uetzand Hošek, 2015).Relying on osteological patterns rather than eye anatomy, Fuhn(1970, 1972) added more skink species with movable lower eyelidsand a transparent disc to Panaspis. Morphological work by Perret(1973, 1975) divided Panaspis species into three groups accordingto general morphology (mabuiform, lacertiform, and sepsinoid).Greer (1974) erected the genus Afroablepharus to accommodateAfrican skinks with an ablepharine eye, and moved all species withmovable lower eyelids and pre-ablepharine eyes to other genera,including semiaquatic species to the genus Cophoscincopus and terrestrial species to the genus Panaspis. As a result, Leptosiaphos wassynonymized with Panaspis based on the movable lower eyelidcharacter, and the only taxon with the pre-ablepharine eye condition was P. cabindae, the type species of Panaspis. Perret (1975)reduced Afroablepharus to a subgenus and described the new subgenus Lacertaspis to accommodate two species (P. reichenowi andP. rohdei) that fitted his lacertiform description from two yearsearlier. Broadley (1989) revised the genera in question andrestricted Panaspis to species residing in African savannas andhaving ablepharine or pre-ablepharine eyes. He then restoredLeptosiaphos to full genus rank for forest and montane grasslandspecies that had a movable lower eyelid. Lastly, he erected a newsubgenus, Perretia, to accommodate a newly described species,Leptosiaphos (Perretia) rhomboidalis, which had distinctive cephaliclepidosis. A recent revision by Schmitz et al. (2005) recognizedAfroablepharus, Lacertaspis, and Leptosiaphos as distinct genera.Although Schmitz et al. (2005) gave Afroablepharus full-genusrank, insufficient sampling did not fully resolve the genus-levelboundaries between Afroablepharus and Panaspis, as only two species each of Afroablepharus and Panaspis were assessed, and samples of the type species of the latter genus (Panaspis cabindae)were not available at that time. The included species of Panaspis(P. breviceps and P. togoensis) were also not ideal representatives,because they have unique morphological characters and habitatpreferences that differ from most remaining members of the genus.This taxonomic arrangement is currently recognized in a recentreptile atlas of South Africa (Bates et al., 2014) and the ReptileDatabase (Uetz and Hošek, 2015). In this study, our objective isto investigate the monophyly of Afroablepharus and Panaspis, andclarify their relationship to closely related African genera, including Lacertaspis and Leptosiaphos. Afroablepharus wahlbergi is themost common and widespread snake-eyed skink in sub-SaharanAfrica, but its distribution is disjunct and poorly known (Branch,1998; Fuhn, 1970; Spawls et al., 2002). Greer (1974) designatedA. wahlbergi as the type species of Afroablepharus. The type localitywas vaguely defined by Smith (1849)—as ‘‘country to the eastwardof the Cape Colony,” but it is likely to be in the southeastern part ofKwaZulu-Natal (Broadley and Howell, 1991). The species has beenreported from mainly southern and eastern African countries fromSouth Africa to Kenya, and even Namibia (Fuhn, 1970; Jacobsenand Broadley, 2000; Spawls et al., 2002). Other sub-Saharan Africanendemics are known to have a similar widespread distributionover savanna and/or woodland habitats, including birds (Voelkeret al., 2012), anurans (Evans et al., 2015; Zimkus et al., 2010), mammals (Gaubert et al., 2005), insects (Simard et al., 2009), and otherskinks (Portik and Bauer, 2012).Herein, we examine evolutionary relationships of skinks in thegenera Panaspis and Afroablepharus. We follow the General LineageConcept (de Queiroz, 1998, 2007; de Queiroz and Gauthier, 1990),which recognizes species as separately evolving lineages. With thisspecies concept, we reject the use of subspecies as natural groupsand use molecular data sets to identify separately evolving species.Our concatenated analyses are used to address the following questions: (1) Are Afroablepharus and Panaspis distinct, reciprocallymonophyletic lineages? (2) What is the extent of cryptic speciationwithin the Afroablepharus wahlbergi complex? (3) When didAfroablepharus/Panaspis species diversify? and (4) Can diversification of Afroablepharus/Panaspis species be linked to climatic andbiogeographic events?2. Materials and methods2.1. Taxon samplingSpecimens of the genera Panaspis and Afroablepharus were collected from multiple localities in sub-Saharan Africa, and 76 samples were sequenced (Table 1, Fig. 1). Additional comparativematerial was obtained from collections listed by Sabaj Pérez(2013). We generated 75 sequences of 16S, 70 of cyt b, 65 of PDC,and 41 of RAG1. Two species of Trachylepis, one species of Typhlosaurus (Scincidae) and Cordylus marunguensis (Cordylidae) wereused as outgroups to root the trees. Additional sequences of closelyrelated genera (Lacertaspis, Leptosiaphos, and Mochlus) were alsosequenced or included from GenBank (Table 1).2.2. PCR amplification and sequencingThe DNA of alcohol-preserved muscle or liver tissue sampleswas extracted using the Qiagen DNeasy Blood and Tissue Kit(Valencia, CA), or the IBI DNA Extraction Kit (Shelton Scientific,Peosta, IA). Two mitochondrial (16S and cyt b) and two nuclear(PDC and RAG1) genes were amplified (Table 2) in 25 lL PCRs, withan initial denaturing temperature of 95 C for 2 min, followed bydenaturation at 95 C for 35 seconds (s), annealing at 50 C for35 s, and extension at 72 C for 95 s with 4 s added to the extensionper cycle for 32 or 34 cycles (for mitochondrial or nuclear genes,respectively). The PCR amplicons were visualized with a 1.5% agarose gel with SYBRsafe gel stain (Invitrogen, Carlsbad, CA), and theseproducts were purified with Agencourt AMPure XP magnetic bead

411M.F. Medina et al. / Molecular Phylogenetics and Evolution 100 (2016) 409–423Table 1Field numbers and localities for specimens used in genetic analyses. DRC Democratic Republic of Congo, E east, Moz Mozambique, N north, NW northwest,SW southwest, S south, SA South Africa.SpeciesField numberCollection numberLocality16Scyt bPDCRAG1Cordylus marunguensisTrachylepis megaluraTrachylepis striataTyphlosaurus brainiTyphlosaurus brainiLacertaspis chriswildiLacertaspis gemmiventrisLacertaspis gemmiventrisLacertaspis reichenowiLacertaspis rohdeiLeptosiaphos blochmanniLeptosiaphos koutouiLeptosiaphos meleagrisLeptosiaphos sp.Leptosiaphos sp.Mochlus aferAfroablepharus africanusAfroablepharus africanusAfroablepharus africanusAfroablepharus annobonensisAfroablepharus annobonensisPanaspis brevicepsPanaspis brevicepsPanaspis brevicepsPanaspis brevicepsPanaspis cabindaePanaspis cabindaePanaspis cabindaePanaspis cabindaePanaspis cabindaePanaspis cabindaePanaspis cabindaePanaspis cabindaeAfroablepharus maculicollisAfroablepharus maculicollisAfroablepharus maculicollisAfroablepharus maculicollisAfroablepharus maculicollisAfroablepharus maculicollisAfroablepharus sp. EthiopiaAfroablepharus sp. Katanga 1Afroablepharus sp. Katanga 1Afroablepharus sp. Katanga 2Afroablepharus sp. Katanga 2Afroablepharus sp. Katanga 2Afroablepharus sp. Katanga 2Afroablepharus sp. Katanga 2Afroablepharus sp. LimpopoAfroablepharus sp. LimpopoAfroablepharus sp. MalawiAfroablepharus sp. MalawiAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. MozambiqueAfroablepharus sp. NamibiaAfroablepharus sp. NamibiaAfroablepharus sp. Tanzania 1Afroablepharus sp. Tanzania 1EBG 2993EBG 1409EBG 1407AMB 6338AMB 6340–RCD 13251RCD 13255E56.12–EBG 1610–ELI 2844––E56.17–Pm3E62.17An15An9ELI 558MM 106MM 105–WRB 804PM 050PM 049WRB 810ANG 21ELI 1722ANL 52MBUR 2128ANG 421MBUR 02843MBUR 02848MCZF 38848MCZF 38790MCZF 38733TJC 264ELI 294ELI 295WRB 575WRB 576JHK 26WRB 0047WRBNimb083MCZ-A 27176MCZ-A 27177WRB 568WRB 570WC 1251WC 1249WC 1169WC 1186SVN 693WRB 886WC 1358ENI 038WC 1051WC 1067WC 1133WC 1161WRB 855WRB 856WC 1317WC 1404ENI 037DMP 187AMB 7634WRB 567WRB 0021WRB 0026UTEP 20374UTEP 21195UTEP 21172CAS 214579CAS 214581ZFMK 75735CAS 207854CAS 207858–ZFMK 75382UTEP 21177MNHN 2001.0697UTEP 21178ZFMK 69552ZFMK 75381ZFMK 54317Uncatalogued–BMNH, Uncatalogued––UTEP 21176ZFMK 87663ZFMK 87662ZFMK 75380PEM R20256UncataloguedUncataloguedPEM R21594PEM R19467UTEP 21173MTD 48612UncataloguedPEM R20475UncataloguedUncataloguedCAS 234188CAS 234135CAS 234099–UTEP 21174UTEP 21175PEM R17454PEM R17455UncataloguedPEM R20327––CAS 248791PEM R20247PEM R20800PEM R20561No voucherPEM R20565PEM R20566–PEM R20591UncataloguedUncataloguedNo voucherPEM R20557No voucherPEM R20558PEM R20569PEM R20576UncataloguedUncataloguedUncataloguedMVZ 266148MCZ R183767Uncatalogued––Pepa, Katanga, DRCLwiro, South Kivu, DRCLwiro, South Kivu, DRCRooibank, Erongo Region, NamibiaRooibank, Erongo Region, NamibiaTchabal Mbabo, CameroonBioko Island, Equatorial GuineaBioko Island, Equatorial Guinea–Mt. Nlonako, CameroonBichaka, South Kivu, DRCMeiganga, Adamaoua Plateau, CameroonRwenzori Mountains National Park, UgandaMt. Nlonako, CameroonMt. Nlonako, CameroonKiyawetanga, KenyaPríncipe, Gulf of GuineaMontalegre, Príncipe, Gulf of GuineaPríncipe, Gulf of GuineaAnnobon, Gulf of GuineaAnnobon, Gulf of GuineaByonga, South Kivu, DRCMawne, CameroonMawne, CameroonMt. Nlonako, CameroonSoyo, NW AngolaLuango-Nzambi, Bas-Congo, DRCLuango-Nzambi, Bas-Congo, DRCRiverine Forest, Bengo, AngolaLagoa Carumbo, AngolaBombo-Lumene Reserve, Kinshasa, DRCKimpa Vita Uni Campus, Uíge, N AngolaS Leba Pass, Huila District, SW AngolaBenero Campsite, near Jamba, AngolaPhalaborwa, Limpopo, SAPhalaborwa, Limpopo, SAFarm Nooitgedacht, Limpopo Province, SAFarm Vrienden, Limpopo Province, SAFarm Vrienden, Limpopo Province, SAOromia, western EthiopiaMulongo, Katanga, DRCMulongo, Katanga, DRCKalakundi Copper Mine, S Katanga, DRCKalakundi Copper Mine, S Katanga, DRCKisanfu Camp, Katanga, DRCFungurume Camp, Katanga, DRCNW ZambiaHoedspruit, Limpopo, SAHoedspruit, Limpopo, SASombani Trail, Mt. Mulanje, MalawiLikabula Station, Mt. Mulanje, MalawiEcofarm, Chemba, MozEcofarm, Chemba, MozBoabab Ore Mine, Masamba, MozBoabab Ore Mine, Masamba, MozGorongosa National Park, MozRuoni Hill S, Tete Province, MozQuiterajo, Cabo Delgado, MozMocimboa da Praia, Cabo Delgado, MozNW of Rapale, Nampula, MozE of Ribuae, Nampula, MozNW of Mecuburi, Nampula, MozRapale, Nampula, MozSyran graphite mine, Balama, MozSyran graphite mine, Balama, MozPemba, Cabo Delgado, MozPemba, Cabo Delgado, MozQuirimbas National Park, MozSerra Jeci, MozSesfontein, NamibiaOtavi, NamibiaArusha, TanzaniaArusha, 8676111111223333444445(continued on next page)

412M.F. Medina et al. / Molecular Phylogenetics and Evolution 100 (2016) 409–423Table 1 (continued)SpeciesField numberCollection numberLocality16Scyt bPDCRAG1Afroablepharus sp. Tanzania 2Afroablepharus sp. Tanzania 2Panaspis togoensisPanaspis togoensisPanaspis togoensisPanaspis togoensisPanaspis togoensisP

Molecular phylogeny of Panaspis and Afroablepharus skinks (Squamata: Scincidae) in the savannas of sub-Saharan Africa Maria F. Medina a , Aaron M. Bauer b , William